Tanshinone IIA prevents Leu27IGF-II-induced cardiomyocyte hypertrophy mediated by estrogen receptor and subsequent Akt activation
Yueh-Shan Wenga, Hsueh-Fang Wangb, Pei-Ying Paic, Gwo-Ping Jongd, Chao-Hung Laid,e, Li-Chin Chungf, Dennis Jine-Yuan Hsiehg, Cecilia Hsuan Dayh, Wei- Wen Kuoi*, Chih-Yang Huanga,j,k*
aGraguate Institute of Chinese Medicine, China Medical University, Taichung, Taiwan
bInstitute of Biomedical Nutrition, Hungkuang University, Taichung , Taiwan
cDivision of Cardiology, China Medical University Hospital, Taichung, Taiwan
dDivision of Cardiology, Armed Force Taichung General Hospital, Taichung, Taiwan
eGraduate Institute of Aging Medicine, China Medical University, Taichung, Taiwan
fDepartment of Hospital and Health Care Administration, Chia Nan University of Pharmacy & Science, Tainan County, Taiwan
g School of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung , Taiwan
hDepartment of Nursing, Mei Ho University, Pingguang Road, Pingtung, Taiwan
iDepartment of Biological Science and Technology, China Medical University, Taichung,Taiwan
jGraduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan
kDepartment of Health and Nutrition Biotechnology, Asia University, Taichung, Taiwan
* These authors contributed equally to this paper.
Correspondence to: Chih-Yang Huang, Ph.D, Graduate Institute of Basic Medical Science and Graduate Institute of Chinese Medicine, China Medical University and Hospital, No. 91, Hsueh-Shih Road, Taichung, 404, Taiwan. Tel: 886-4-2205-3366 ext. 3313, FAX number: 886-4-2207-0465, E-mail: [email protected]
Abstract
IGF-IIR plays important roles as a key regulatorin myocardial pathological hypertrophy and apoptosis, which subsequently lead to heart failure.
Salviamiltiorrhiza Bunge (Danshen) is a traditional Chinese medicinal herb used to treat cardiovascular diseases. Tanshinone IIA is an active compound inDanshen and is structurally similar to 17β-estradiol (E2). However, whether tanshinone IIA improves cardiomyocytesurvival in pathological hypertrophy through estrogen receptor (ER) regulation remains unclear. This study investigates the role of ER signaling in mediating the protective effects of tanshinone IIA on IGF-IIR-induced myocardial hypertrophy. Leu27IGF-II (IGF-II analog) was shown in this study to specifically activate IGF-IIR expression and ICI 182,780 (ICI), an ER antagonist used to investigate tanshinone IIA estrogenic activity. We demonstrated that tanshinone IIA significantly enhanced Aktphosphorylation through ER activation to inhibit Leu27IGF-II-induced calcineurin expression and subsequentNFATc3 nuclear translocation to suppress myocardial hypertrophy. Tanshinone IIA reduced cell size and suppressed ANP and BNP, inhibitinganti-hypertrophic effects induced by Leu27IGF-II. The cardioprotective properties of tanshinone IIA that inhibit Leu27IGF-II-induced cell hypertrophy and promote cell survival were reversed byICI.Furthermore, ICI significantly reduced phospho-Akt, Ly294002 (PI3k inhibitor), and PI3k siRNA significantly reduced the tanshinone IIA-induced protective effect. The above results suggest that tanshinone IIA inhibited cardiomyocyte hypertrophy, which was mediated through ER, by activatingthe PI3k/Akt pathway and inhibiting Leu27IGF-II-induced calcineurin and NFATc3.
Tanshinone IIA exerted strong estrogenic activity and therefore represented a novel selective estrogen receptor modulator that inhibits IGF-IIR signaling to block cardiac hypertrophy.
Key words:Tanshinone IIA; selective estrogen receptor modulator (SERM); IGF-II receptor; Cardiac hypertrophy; calcineurin/NFAT3; p-Akt.
Introduction
Cardiac hypertrophy is an adaptive response to biomechanical stress stimuli,such as hypoxia, hemodynamic overload and myocardial injury, or neurohormone growth factors including angiotensin II (AngII) and Insulin-like growth factors caused by cardiac remodeling such as progression from physiological hypertrophy to pathological hypertrophy . Prolonged cardiac hypertrophy leads to cardiomyocytesapoptosis and is associated with a significantly increased risk for heart failure and malignant arrhythmia. During cardiac hypertrophy, the genes encoding atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and β-muscle myosin heavy chain (β-MHC) are re-expressed . These molecules induce sarcomeres and neurofilamentsremodeling and enhance protein synthesis, which lead to increased muscle-cell size . Accumulating evidence implicates the calcineurin-dependent transcriptional pathway as a mediator of cardiac hypertrophy. Hypertrophic stimuli such as AngII or phenylephrine (PE) induce calcium influx, which activates calcineurin (protein phosphatase 2B). Calcineurin dephosphorylates NFAT3, which then translocates to the nucleus to activate the transcription of genes that encode the proteins involved in hypertrophy .
During embryogenesis, the genes encoding ANP and BNP are expressed in the atrium and ventricle , and their levels increase dramatically in association with cardiachypertrophy. Therefore, along with β-MHC,ANP and BNP provide markers for pathological cardiac hypertrophy.Moreover, plasma BNP levels are used to diagnoseand manage heart failure .The reappearance of ventricular ANP expression in
adults is recognized as a marker for the induction of the embryonic gene program in ventricular hypertrophy. Increased ANP and BNP expression occurs in myocardial infarction animal models , heart failure and hypertrophy , as well as in human heart disease Consequently, increased expression of the gene encoding ANP in adult ventricular tissue is a marker for the inappropriate induction of the embryonic gene program in the development of ventricular hypertrophy .
Insulin-like factor-II (IGF-II) plays an important role in fetoplacental growth throughout gestation, including fetal cell division, differentiation, cell cycle progression and DNA synthesis . IGFs present in plasma are synthesized in the liver.
IGF-II binds to the IGF-II receptor (IGF-IIR) with higher affinity than to the IGF-IR.
IGF-II activates tyrosine kinase and initiates a protein kinase cascade .Spontaneously hypertensive (SHR) rats express high levels of cardiac IGF-II and IGF-IIR and low levels of IGF-I mRNA and protein during fetal and postnatal periods . Previous studies suggested that IGF-IR-dependent signaling inhibition causes IGF-II to bind to the IGF-IIR, which activates the IGF-IIR-dependent signaling pathway and induces cardiomyocytes hypertrophy.
We found that IGF-IIR activation is associated with pathological hypertrophy progression in an abdominal aorta ligation rat model . IGF-II and IGF-IIR activation mediates JNK and ERK activation by AngII, which regulates apoptosis in the H9c2 cardiomyoblast cell line . In our previous studies, using immunohistochemical techniques, we discovered a significant association between IGF-IIR overexpression and myocardial infarction . We also demonstrated that, in H9c2 cells, IGF- II/mannose-6-phosphate-receptor signaling induces cell hypertrophy and the
expression of ANP and BNP via interactions with Gαq and activation of protein kinase C-α . Furthermore, using the IGF-II analog Leu27IGF-II to specifically activate the IGF-IIR , we investigated the role of IGF-II/IGF-IIR activation and its downstream signaling pathway. Our results revealed that Leu27IGF-II induction of calcineurin activity and apoptosis was required for the interaction between the αq polypeptide (Gαq) and IGF-IIR . Therefore, we wished to determine whether suppressing signaling through the IGF-IIR pathway provides a strategy for protecting against cardiac hypertrophy and progression to heart failure.
Danshen(Salviamiltiorrhiza Bunge) protects humans from pathologies such as cardiovascular dysfunction , cancers , and diabetes mellitus . The phytoestrogen tanshinone IIA is the major bioactive constituent of Danshen root, which is used widely in China to treat cardiovascular diseases. Evidence indicates that tanshinone IIA exerts antioxidant , anti-inflammatory , and antiapoptotic effects on the cardiovascular system . Tanshinone IIAis structurally similar to 17β-estradiol (E2) (Figure 1), and the tanshinone IIA content of Danshen is 1.089%. Tanshinone IIA binds to ERα and ERβ and activates gene transcription in estrogen-sensitive tissues and cell lines . Danshen extract significantly enhanced phosphorylated Akt through ERs activation to suppress the IGFIIR apoptotic pathway by Leu27IGF-II-induced calcineurin activation in myocardial cells. . However, the Tanshinone IIA role in inducing ER activation and suppressing IGF2R-induced hypertrophy has never been studied in detail. In this study we examined the anti-hypertrophy effect of tanshinone IIA induced by Leu27IGF-II on H9c2 cardiomyoblast and neonatal rat ventricular myocytes. The role of tanshinone IIA in manipulating the main functions that activate the ER/Akt pathway, namely PI3k/Akt signaling and anti-hypetrophy, has also been
studied in detail with an aim to elucidate the possible signal transduction pathways underlying these actions.
Materials and methods
Chemicals
Tanshinone IIA was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA), and its purity was found to be a minimum of 97% by high-performance liquid chromatography. Leu27IGF-II was purchased from GropepBioreagents Pty Ltd (Australia). The estrogen receptor antagonist (ICI 182 780; ICI) was purchased from TOCRIS (Ellisville, Missouri, USA). The calcineurin inhibitorcyclosporin A(CsA) was purchased from Sigma-Aldrich Chemical Co. Rhodamine–phalloidin was purchased from Molecular Probes (Eugene, Oregon, USA). The Alexa Fluor 488
donkey anti-goat IgG (H+L) was purchased from Invitrogen (Carlsbad, California, USA). We purchased the following from Santa Cruz Biotechnology, Inc. (Santa Cruz, California, USA): antibodies against α-tubulin and β-actin, which were used as a loading control; antibodies against ANP, BNP, calcineurin, phosphorylated NFATc3, NFATc3, Akt, and phosphorylated Akt (ser473); and goat anti-mouse IgG antibody conjugated to horseradish peroxidase, goat anti-rabbit IgG antibody conjugated to horseradish peroxidase, and donkey anti-goat IgG horseradish peroxidase conjugated to horseradish peroxidase.
Cell culture and treatment
The rat cardiomyoblast cell line (H9c2) was purchased from theBioresource Collection and Research Center (BCRC, Taiwan). H9c2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma Aldrich, MO, USA) with 10% fetal bovine serum (HyClone, USA), 100 μg/ml penicillin, 100 μg/mlstreptomycin, 2 mM glutamine, and 1 mM HEPES buffer. The cultures were incubated at 37 °C in a humidified (10%) atmosphere containing 5% CO2. After the cells were deprived of serum (i.e. in serum-free DMEM) for 4 h and treated with different drugs at different concentrations and time points.
Neonatal rat ventricle cardiomyocyte culture
Neonatal rat cardiomyocytes were isolated and cultured using the Neonatal Cardiomyocyte Isolation System Kit according to the manufacturer’s instructions (Cellutron Life Technology, Highland Park, New Jersey, USA). Briefly, hearts from 1- to 2-day-old Sprague-Dawley rats were removed, the ventricles were pooled, and the ventricular cells were dispersed using the kit’s digestion solution in a 37 °C incubator. To select cardiac fibroblasts, cells were centrifuged and cell pellets
resuspended in D3 buffer (Cellutron Life Technology) and plated for 2 h in an uncoated plate at 37 °C in a 5% CO2 incubator. Prepare 4 ml SureCoat solution was used to coat each 10 cm plate for at least 1 h in a 37 °C incubator. The unattached cells (ventricular cardiomyocytes) were transferred to coated plates with NS medium (supplemented with 10% fetal bovine serum). Ventricular cardiomyocytes were isolated and cultured in DMEM containing 10% fetal bovine serum, 100 μg/ml penicillin, 100 μg/ml streptomycin, and 2 mM glutamine. After 3-4 days, cells were incubated in serum-free essential medium overnight before treatment with the indicated agents.
Cell viability assay
Cell viability was determined using 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) that isconverted to blue formazan by mitochondrial succinatedehydrogenase, specifically in living cells. H9c2 cardiomyoblast cells were seeded into 24-well plates and subjected to various treatments. Tanshinone IIA was added to the wells at different concentrations (0, 10 and100 nM) for 6, 12, and 24 h.
The culture medium was replaced with 100 μl of MTT solution(5 mg/ml stock solution in phosphate buffered saline (PBS) diluted with culture mediumto a final concentration of 0.5 mg/ml). After 4h incubation at 37 °C,the medium was removed, and the formazan was solubilizedin 500 μl of dimethyl sulfoxide (DMSO) and its absorbance at 570 nm was determined using an automated microplate reader (Molecular Device Spec 384).
Western blot analysis
To isolate total proteins, cultured H9c2 cardiomyoblast cells were washed with cold PBS and resuspended in lysis buffer (50 mM Tris, pH 7.5, 0.5 M NaCl, 1.0 mM
EDTA, pH 7.5, 10% glycerol, 1 mM BME, 1% IGEPAL-630) mixed with proteinase inhibitor cocktail (Roche Molecular Biochemicals). After incubation for 30 min on ice, the lysate was centrifuged at 12,000 g for 15 min at 4 °C, and the supernatant was collected for western blot analysis. The protein concentration of the supernatant was determined using the Bradford method. Samples containing equal amounts of protein (40 μg) were loaded and subjected to western blotting analysis. Briefly, the proteins were separated using 12% SDS-PAGE and transferred onto a PVDF membrane (Millipore, Bedford, MA, USA). Membranes were treated with blocking buffer (5%
nonfat dry milk, 20 mMTris-HCl, pH 7.6, 150 mMNaCl, and 0.1% Tween 20) for at least 1 h at room temperature. Membranes were incubated with primary antibodies in the above solution on an orbital shaker at 4 °C overnight. Following incubation with primary antibodies, the membranes were incubated with horseradish peroxidase- conjugated secondary antibodies (anti-rabbit, anti-mouse, or anti-goat IgG; Santa Cruz Biotechnology, Inc., California, USA).
Total RNA extraction, reverse transcription, and polymerase chain reaction (PCR) amplification
Total RNA was extracted using the Zymo mini RNA isolation kit (Zymo Research) to isolate RNA from 103–105 cells/sample (Zymo Research, Orange, CA). An aliquot of total RNA (0.5 μg) was reverse-transcribed using 0.5 μM oligo(dT) primers in a solution (50 μl) containing 75 mMKCl, 50 mMTris-HCl (pH 8.3), 3 mM MgCl2, 10 mM DTT, 10 U RNase inhibitor (Promega, Madison, Wisconsin, USA), 0.8 mM total dNTPs, and 200 U of Moloney murine leukemia virus reverse transcriptase (Promega). The sample was incubated at 42 °C for 1 h and at 99 °C for 5 min before
chilling on ice for 10 min. The product (2 μl) was diluted with PCR buffer (50 mMKCl, 10 mMTris-HCl, and 2 mM MgCl2) to a final volume of 50 μl containing 0.5 μMdNTPs (final concentration, 0.8 mM) and 0.5 U TaqDNA polymerase. For each experiment, up to 32 cycles were performed to avoid reaching the PCR plateau values.
PCR amplification was initiated with a hot start (5 min at 95 °C), and the samples were then subjected to 32 cycles at 95 °C for 1 min, 58 °C for 1 min, and 72 °C for 2 min. The annealing temperature for the ANP and IGF-IIR primers was 60 °C and the annealing temperature for the GAPDH primers was 54 °C. This was followed by a final extension step at 72 °C for 20 min.The PCR products were analyzed using 1.2%
agarose gelelectrophoresis, and the amplicons were visualized using a KodakScientific 1D Imaging System (Eastman Kodak Company, New Haven,CT).
The primers used wereas follows: rat ANP forward primer 5′-
TGCCGGTAGAAGATGAGGTC-3′ and reverse primer 5′-
ATTCACCACCTCTCAGTGGC-3′; rat IGF-IIR forwardprimer 5′- ATGCACCGTGCGGAATGGAAGCTCG-3′ and reverse primer 5′- TCACCTGGCAGATGTTGGCACCGGA-3′; and rat GAPDH forward primer 5′-
GGGTGTGAACCACGAGAAAT-3′ and reverse primer 5′-
CCACAGTCTTCTGAGTGGCA-3′ (MDBio, Taipei, Taiwan).
Immunofluorescence analysis of actin
Cardiomyocytes were fixed with 4% paraformaldehyde at room temperature for 30 min. Cells were washed four times with cold PBS and permeabilized with 0.1%
Triton X-100 for 10 min at 4°C. Nonspecific binding of the fixed cells was blocked with PBS containing 2% bovine serum albumin at room temperature for 30 min. Actin filaments were visualized using rhodamine-labeled phalloidin. Cells were examined
and photographed using a Zeiss Axioskop fluorescence microscope. The cells were viewed at a magnification of 400× and analyzed using Zeiss AxioVision software to determine their surface areas. Sixty cells from each experiment were analyzed, and three independent experiments were performed using each treatment condition.
Immunofluorescence analysis
Cardiomyocytes were fixed with 4% paraformaldehyde at 4°C for 30 min. Cells were washed four times with ice-cold PBS and permeabilized with 0.1% Triton X-100 for 10 min at 4°C. Nonspecific binding of the antibody by the fixed cells was blocked using PBS containing 2% bovine serum albumin at room temperature for 30 min, which was followed by incubation with an anti-NFAT3 antibody overnight at 4 °C.
After the cells were washed, they were incubated with anti-goat IgG labeled with Alexa Fluor® 488 at 37°C for 1 h. The samples were then stained with 1 μg/mlof 4′,6- diamidino-2-phenylindole dihydrochloride (DAPI, Roche) for 10 min to detect the cell nucleus (blue stain) using ultraviolet light. Cells were stained with Alexa Fluor® 488-labeled antibody alone as the negative control. After staining with the antibody, fluorescence was visualized using a Zeiss Axioskop fluorescence microscope coupled to an image analysis system.
Short interfering RNA (siRNA) transfection
PI3k siRNAs (ON-TARGETplusSMARTpool rat Pik3r1; Gene ID: 25513) were purchased from Thermo Scientific (MA, USA). PI3k siRNAs and non-targeting siRNAs were transfected using the TurboFect™ in vitro Transfection Reagent (Fermentas) according to the manufacturer’s instructions. The final concentration of PI3k siRNAs for transfection was 100 nM. The transfected cells were washed, incubated in fresh culture media, and exposed to the agents, as indicated in the results
section.
Statistical analysis
Results have been expressed as the mean ±standard deviation (SD) of the mean of three independent experiments conducted in triplicate. Data were analyzed using the SPSS 12.0 software with one-way analysis of variance (one-way ANOVA) followed by Scheffe’s test.
Results
Leu27IGF-II induces upregulation of the expression of ANP and BNP in myocardial cells
ANP and BNP are the markers of pathological cardiac hypertrophy. To better understand whether the ANP and BNP expressioninduced by Leu27IGF-II treatment, we performed western blotting analysis of H9c2cells treated with Leu27IGF-II (0.1,1 and 10 nM) (Fig. 2). The results showed that Leu27IGF-II increased ANP and BNP expression in a dose-dependent manner.
Tanshinone IIAattenuates hypertrophy of H9c2 cardiomyoblasts induced by Leu27IGF-II
To determine the tanshinone IIAeffects on Leu27IGF-II-induced hypertrophy in H9c2 cardiomyoblasts, we treated the cells with the ER antagonist ICI. Actin fiber immunofluorescence detection was used to measure cellular areas. After incubation with Leu27IGF-II (10 nM) for 24 h, a marked increase in actin filament organization was observed, which was partially inhibited by tanshinone IIA (Fig. 3). The H9c2 cell areas increased significantly by a factor of approximately 2.2 in the presence of Leu27IGF-II for 24 h, compared with the control. Tanshinone IIA (10 and 100 nM) significantly reduced this effect by factors of approximately 0.96 and 0.94, respectively. However, ICI restored the antihypertrophic effect induced by tanshinone IIA and the cell size increased significantly by a factor of 1.6 compared with the control. These findings suggest that treatment with tanshinone IIA dramatically inhibited H9c2 cardiomyoblast hypertrophy induced by Leu27IGF-II and further
suggests that the antihypertrophic effect of tanshinone IIA was mediated through ERs.
Tanshinone IIA inhibits the ANP and BNP expression induction by Leu27-IGF-II in myocardial cells
To further investigate whethertanshinone IIA inhibited Leu27IGF-II-induced pathological hypertrophy inmyocardial cells, we determined ANP and BNP expression, which are markers of pathological hypertrophy, using western blotting and RT-PCR analyses.The ANP and BNP induction in response to Leu27IGF-II (10 nM) was inhibited to different extents by 10 and 100 nMtanshinone IIA.Compared with the findings from the control, the ANP and BNP levels increased significantly in myocardial cells treated with Leu27IGF-II alone for 24 h (Fig. 4A). Tanshinone IIA significantlyinhibited the Leu27IGF-II-induced ANP and BNP expression. The inhibitory effect was attenuated by ICI. To further confirm the western blotting analyses results, the tanshinone IIA effects on the ANP and IGF-IIR mRNAs levels were evaluated in Leu27IGF-II-treated neonatal cardiomyocytes.Tanshinone IIA treatment significantly decreased the Leu27IGF-II-induced ANP and IGF-IIRmRNAs expression (Fig. 4B).ICI inhibited the tanshinone IIA (10 and 100 nM) ability to inhibit ANP and IGF-II mRNAs expression in neonatal cardiomyocytes. These results suggest that tanshinone IIA counteracted the increase in ANP and BNP levels induced by Leu27IGF-II and that this effect might be mediated through ERs.
Calcineurin mediates Leu27IGF-II-induced myocardial cell hypertrophy
To determine whether Leu27IGF-II-induced myocardial hypertrophy was mediated through calcineurin/NFAT3 signaling, cyclosporine A (CsA, calcineurin
inhibitor) and tanshinone IIA were used to inhibit the calcineurin/NFAT3 signaling pathways after Leu27IGF-IIadministration. The western blotting analysis presented in Fig. 5A. shows that CsA significantly inhibited Leu27IGF-II-induced NFATc3 and calcineurin expression. Furthermore, pretreatment with CsA and tanshinone IIA significantly inhibited the Leu27IGF-II-induced increase in ANP and BNP expression (Fig. 5B.). These results suggest that calcineurin modulated the Leu27IGF-II-induced ANP and BNP expression in myocardial cells and that tanshinone IIA inhibited calcineurin signaling in a manner similar to CsA.
Tanshinone IIA inhibits Leu27IGF-II-induced NFAT3 nuclear translocation in H9c2 cardiomyoblast cells
We next investigated the possibility that tanshinone IIA inhibited the IGF-IIR- induced translocation of NFAT3 to the nucleus in myocardial cells. The subcellular localization of NFAT3 was analyzed using immunofluorescence microscopy and western blotting analyses. Leu27IGF-II treatment induced the translocation of NFAT3 from the cytosol to the nucleus, which was completely inhibited by tanshinone IIA (Fig. 6A.). However, the inhibitory effect of tanshinone IIA significantly decreased when H9c2 cells were treated with 2 μM ICI (Fig. 6A.). To further investigate whether tanshinone IIA inhibited the Leu27IGF-II-induced calcineurin/NF-AT3 hypertrophic signaling pathway in H9c2 cells, the NFAT3 and calcineurin levels were analyzed using western blotting. The NFAT3 and calcineurin levels increased in the presence of 10 nM Leu27IGF-II but were significantly decreased by 10 and 100 nMtanshinone IIA, compared with the results obtained for cells treated with Leu27IGF-II alone (Fig. 6B.). Moreover, Leu27IGF-II effects inhibitionby tanshinone
IIA was totally reversed by ICI. These results suggest that tanshinone IIA prevented the activation of calcineurin/NFAT3 signaling by Leu27IGF-II and that the H9c2 cardiomyoblastshypertrophy was modulated through ERs.
Tanshinone IIA inhibits Leu27IGF-II-induced H9c2 cardiomyoblast hypertrophy through activation of the PI3k/Akt pathway
To analyze the tanshinone IIA activated ER mechanisms that might prevent cardio hypertrophy in more detail we assessed whether tanshinone IIA mediated Akt phosphorylation to modulate hypertrophy, at least in part, using“non-genomic”
pathways. It is well known that the ER interacts with the PI3K/Akt pathway in myocytes. The ICI was applied to inhibit ER activity. Treating H9c2 cells with 2 µM ICI for 1 h significantly inhibited phospho-Akt expression in the presence of 10 and 100 nMtanshinone IIA (Fig. 7A.). This result suggests thatthe PI3k/Akt signaling pathway played a role in ER downstream signaling activation.
To further confirm the functional role of Akt in mediating the tanshinone IIA ability to prevent H9c2 cell hypertrophy, we applied the PI3-kinase inhibitor Ly294002 to suppress Akt downstream activation. Ly294002 significantly suppressed tanshinone IIA inhibitory effects on Leu27IGF-II-induced calcineurin, NFATc3, and BNP expression (Fig. 7B).Tanshinone IIA inhibited the BNP expression induced by Leu27IGF-II, which was attenuated in the presence of Ly294002, suggesting that the cardioprotective properties of tanshinone IIA were mediated through the PI3k/Aktsignaling pathway.
PI3k siRNA inhibits the cardioprotective effects of tanshinone IIA in Leu227IGF-II-
treated neonatal rat ventricular myocytes by inhibiting NFAT3 translocation to the nucleus
To further confirm the functional role of Akt in mediating tanshinone IIA protective effects on primary neonatal rat ventricular myocytes, PI3k and control siRNAs were used to transfect neonatal rat ventricular myocytes. Thirty-six hours after siRNA transfection, neonatal rat ventricular myocyteswere harvested and the PI3k levels were assessed using western blotting analysis with PI3k specific antibody.
Transfection with siRNAs significantly down-regulated PI3k expression and blocked downstream Aktactivation in the presence of tanshinone IIA (100 nM) (Fig. 8 A).Thirty-six hours after siRNA transfection, neonatal rat ventricular myocytes were exposed to 100 nMtanshinone IIA for 2 h before the addition of Leu27IGF-II.
Immunofluorescence microscopy was used to determine NFATc3 subcellular localization. The results confirmed that tanshinone IIA inhibited Leu27IGF-II-induced NFATc3 translocation to the nucleus in myocardial cells, which in turn was inhibited by PI3k siRNA (Fig. 8B). Furthermore, the actin staining results showed that PI3k siRNA inhibited the tanshinone IIA antihypertrophic effects by inducing IGF-IIR expression in ventricular cardiomyocytes (Fig. 8C). These findings demonstrate that tanshinone IIA inhibited Leu27IGF-II-induced cell hypertrophy and promoted cell survival through the PI3k/Akt signaling pathway.
Discussion
Tanshinone IIA is the most abundant and well-studied lipophilic constituent of Danshen and is structurally similar to 17β-estradiol.Tanshinone IIA targets multiple
regulatory molecules such as transcription factors , scavenger receptors , ion
channels , growth factors , inflammatory mediators , microRNAs , and antioxidants . The results from a clinical study suggested that tanshinone IIA attenuates
atherosclerotic inflammatory reactions and thereby protects the heart against injury.However, the tanshinone IIA effect in Leu27IGF-II-induced cardiac
hypertrophy and the related signaling mechanisms remain to be clarified. Therefore, to address this gap in our knowledge,we used tanshinone IIA in the present study to investigate the mechanism that mediates the detrimental effects of Leu27IGF-II- activated IGF-IIR signaling in H9c2 cardiomyoblasts and neonatal rat ventricular myocytes and assess whether the protective role of tanshinone IIA is mediated by ERs.The results from this study indicate that Leu27IGF-II-induced hypertrophy in cardiomyocytes was significantly mitigated by tanshinone IIA. We demonstrated that this mechanism involved the inhibition of hypertrophy-related IGF-IIR signaling, calcineurin activation, NFAT3 translocation to the nucleus and the increase in ANP and BNP expression. Tanshinone IIA upregulated the phosphorylation of Akt and the tanshinone IIA effects were reversed by the ERs antagonist (ICI), indicating that tanshinone IIA exerted its protective effects through ERs. We conclude that tanshinone IIA activates ERs and upregulates Akt phosphorylation.
Cardiac remodeling induced by mechanical stretching or neurohumoral growth factors is an adaptive process that causes cardiac hypertrophy, which is an important risk factor for mortality and morbidity . Although initially beneficial, sustained cardiac hypertrophy leads to decompensation and ventricular dilation, contractile dysfunction and heart failure. In the present study we first determined that tanshinone IIA attenuated H9c2 cell hypertrophy induced by Leu27IGF-II.Next we showed that
tanshinone IIA down regulated the expression of molecular markers for the
hypertrophy pathway. Specifically, we observed increases in cell size (Fig. 3) and in the mRNA and protein expression levels of pathological hypertrophy markers (ANP and BNP) (Fig. 4B and 5) induced by Leu27IGF-II. We found that tanshinone IIA suppressed the Leu27IGF-II hypertrophic effect and inhibited signaling through its downstream modulator calcineurin (Fig. 4), suggesting that tanshinone IIA prevented cardiomyoblast cell hypertrophy in response to IGF-IIR signaling pathway activation by Leu27IGF-II. The results indicated that tanshinone IIA decreased cardiac pressure.
The tanshinone IIA effect is similar phytoestrogen resveratrol effect indecreasing cardiac ANP mRNA expression andpreventing cardiac hypertrophy(Bialaet al.,2010).
IGF-IIR signaling pathway activation stimulates cardiomyocyte hypertrophy through cross-talk with the small G-protein Gαq that regulates the
cardiomyocytesphenotype by activating specific intracellular signaling cascades . Evidence indicates that IGF-IIR overexpression and signaling pathway activation in myocardial infarction tissue activates apoptosis , hypertrophy , and fibrosis, which correlates with the progression of heart disease. IGF-IIR is overexpressed in H9c2 cells treated with TNF-α, LPS, ANGII, or ionomycin in SHR hearts as a function of time and in the myocardial infarction tissue of humans . IGF-IIR activation is
associated with pathological hypertrophy progression in an abdominal aorta rat model . Consistent with these findings, we show here that Leu27IGF-II induced an increase in the IGF-IIR mRNA levels, indicating that Leu27IGF-II binding to IGF-IIR initiated calcineurin-induced hypertrophy and NFAT3 translocation to the nucleus (Fig. 4).
Additionally, we demonstrated that tanshinone IIA attenuated IGF-IIR activation and calcineurin/NFAT3 signaling pathways by Leu27IGF-II, leading to a decrease in
myocardial cell hypertrophy (Fig. 5 and 6).
Numerous intracellular signaling pathways are implicated in the transduction of hypertrophic stimuli. The cell surface receptors AngII, PE and ET-1 mediate calcium influx, which activates protein kinase C and calcineurin . Calcineurin is a phosphatase specific for serine and threonine residues and is activated by a sustained increase in calcium levels. Many studies have implicated calcineurin activity in the development of cardiac hypertrophy . Calcineurin activation by phosphorylation results in the dephosphorylation of NFAT3, which then translocates to the nucleus via a combinatorial mechanism involving its direct interaction with GATA4 and the subsequent induction of genes that are expressed in fetal cardiac tissue . In calcneurin or NFAT3 gene activation, transgenic mice appeared to develop cardiac hypertrophy, dilated cardiomyopathy associated with renal interstitial fibrosis and congestive heart failure .
The immunosuppressantsCsA and FK506 suppress the immune response by inhibiting the ability of calcineurin to activate NFAT transcription factors . In primary cardiomyocytes, treatment with CsA and FK506 inhibits the morphologic and
molecular responses to hypertrophy induced by AngII and PE, indicating that calcineurin is a component of the signaling pathways involved in inducing hypertrophy. Moreover, CsAadministration prevents cardiac hypertrophy and the associated pathology in calcineurin transgenic mice . The present study shows that when IGF-IIR is activated by specific binding to Leu27IGF-II, calcineurin is activated. This induces cellular hypertrophy because NFATc3 translocates to the nucleus to induce the transcription of genes that normally encode ANP and BNP only
during embryogenesis. Our data demonstrate that Leu27IGF-II induced an increase in calcineurin levels (Figs. 4A and 5B) and that tanshinone IIA and CsA significantly inhibited calcineurin activation and translocation of NF-ATc3 to the nucleus (Fig.
4A), and the subsequent down regulation of ANP and BNP (Fig. 4B) in H9c2 cells exposed to Leu27IGF-II. Furthermore, we present evidence that calcineurin modulates ANP and BNP expression induced by Leu27IGF-II in myocardial cells and that tanshinone IIA inhibited signaling through calcineurin in a manner similar to that of CsA.Tanshinone IIA attenuated IGF-IIR signaling induced by Leu27IGF-II,
suppressed calcineurin activity and down regulated its downstream modulator NFAT, which contributes to pathological hypertrophy.
The estrogen effects are mediated primarily through ERα or ERβ, or both.These are expressed in the heart and mediate cardio protection.Accumulating evidence demonstrates that ERβ is involvedin mediating E2-induced cardio protection in female individuals, following ischemia/reperfusion injury combined with the isoproterenol-induced hypercontractile condition .We discovered that the
cardioprotective effect of E2 and ERα involves Akt activation by inhibiting JNK1/2 and thus down regulating LPS-induced TNF-α activity . Furthermore, 17β-estradiol (E2) suppresses cardiomyocyte apoptosis via (PI3k)/Akt pathway activation
duringmyocardial infarction .Hormonal replacement therapy (HRT) is implicated in the significantly increased incidence of pulmonary embolism, coronary artery disease and breast cancer . SERMs exhibit either estrogen agonist or antagonist activity in a tissue-specific manner . The SERMs structure and their specific target tissue are closely related .For example, whether a molecule acts as an estrogen antagonist or agonist is determined by the DNA binding response specificity elements in the cells of
specific target tissues . SERMs are used to prevent HRT side effects .
Danshen extract is used clinically in China to treat menopausal syndrome.We discovered in this study that tanshinone IIAserves as an effective SERM.Tanshinone IIA inhibits the proliferation of ER-positive and ER-negative human breast cancer cells (MCF-7 and MDA-MB-231, respectively)in vitro and inhibits the growth ofER- negative breast cancer cell xenografts in nude mice by down regulatingthe gene expression encoding P53 and BCL-2, which mediate apoptosis.Tanshinone IIA is a significantly stronger inhibitor of ER-positive breast cell proliferation in vitro and in vivo than tamoxifen .Tanshinone IIA is an effective estrogen-dependent breast cancer cell antagonist and mimics the beneficial action of estrogen on bone density and prevents osteoclast differentiation . In vivo studies have demonstrated that tanshinone IIA treatment prevents ovariectomy-induced cancellous bone loss in rats through inhibition of elevated bone reabsorption and does not affect uterine weight, while protecting against bone loss induced by estrogen depletion . Tanshinone IIA activates the transcription of genes that encode ERα and ERβ in transfected HeLa cells with a lower affinity than E2 .In LPS-induced RAW264.7 cells, tanshinone IIA exerts anti- inflammatory effects by inhibiting the expression of genes that encodeiNOS, NO production and the expression of inflammatory cytokines (IL-1β, IL-6, and TNF-α) via an ER-dependent pathway . In primary cardiac microvascular endothelial cells, tanshinone IIA induces vasorelaxation and activates the ER signaling pathway, leading to increased expression of the gene that encodes endothelial NOS, nitric oxide production, ERK1/2 phosphorylation, and Ca2+ mobilization . Taken together, the studies described above prove that tanshinone IIA exerts strong estrogenic activity in endothelial cells, immune cells and HeLa cells.However, our study was initiated to
determine whether tanshinone IIA exerts estrogenic activity in myocardial cells. Here, we show that tanshinone IIA acts as a phytoestrogen complex and exerts anti-
hypertrophy effects induced by Leu27IGF-II and blocks the cardiomyoblast
hypertrophic responses such as cell size increase, ANP, BNP, calcineurin and NFAT3 expression. All of the suppressive effects of tanshinone IIA on cardiomyocytes were apparently mediated through ER activation, which were largely reversed by ICI treatment (Fig. 3, 5 and 6). Therefore, tanshinone IIA supplementation protected cardiomyocytes against Leu27IGF-II-induced hypertrophy.These results may explain the effectiveness and mechanisms of S. miltiorrhiza, which is traditionally used as an herbal medication for treating cardiovascular disease.
Akt is a protein serine/threonine kinase and a major mediator of the downstream effects of PI3k signaling, which coordinates intracellular signaling and the regulation of cell proliferation and survival.Akt is a critical therapeutic target for the treatment of cardiac disease. Activation of the PI3k/Akt signaling pathway protects the
myocardium from myocardial injury and prevents cardiacmyocyte apoptosis , andER activates PI3k/Akt signaling to protect against myocardial injury . In diabetic rats with ischemia/reperfusion injury,tanshinone IIA acts through the PI3k/Akt pathways to reduce infarct size, which improves cardiac function . In our present study, tanshinone IIA activated Akt phosphorylation induced by signaling through the ERs and
prevented NFAT3dephosphorylation in cardiomyocytes (Fig. 7). These findings were further supported by our demonstration that PI3k siRNA inhibited the tanshinone IIA–
mediated protective effects on cardiomyocyte hypertrophy and NFAT3 translocation (Fig. 8).
In summary, we show that tanshinone IIA inhibited Leu27IGF-II-induced pathological hypertrophy mediated by ERs through PI3k/Akt signaling pathway activation, which inhibits calcineurin and NFATc3 expression (Fig. 9). Taken together, our results suggest that tanshinone IIA plays an important role in inhibiting calcineurin/NFAT3 to prevent pathological hypertrophy. These results strongly suggest that S.miltiorrhiza is effective in preventing the development of cardiac hypertrophy in postmenopausal women.
5. Acknowledgment
This study is supported in part by Taiwan Ministry of Health and Welfare Clinical Trial and Research Center of Excellence (MOHW104-TDU-B-212-113002).
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Figure legends
Fig. 1. The chemical structure of tanshinone IIA.
Fig. 2. The pathologic hypertrophy markers ANP and BNP were induced by
Leu27IGF-II in myocardiac cells. H9c2 cardiomyoblast cells were cultured in DMEM containing Leu27IGF-II at the indicated concentrations (0.1, 1, and 10 nM) for 24 h.
Total cell extract proteins were separated using 12% SDS-PAGE, transferred to PVDF membranes and analyzed using western blot with antibodies against proteins.
Equal loadswere assessed with an anti-β-actin antibody. Results are shown in mean ± S.E.M (n = 3). *=P< 0.05, ** =P< 0.01 represent significant differences from the control group.
Fig. 3.Tanshinone IIA inhibited cardiomyocyte hypertrophy induced by Leu27IGF-II.
Cardiomyocytes were pretreated with ICI 182,780 (2 μM) for 1 h, followed by incubation with tanshinone IIA (10 nM and 100 nM) for 2 h in the presence of Leu27IGF-II (10 nM) administration for 24 h and then harvested.The tanshinone IIA inhibitory effect on Leu27IGF-II-induced hypertrophy in H9c2 cardiomyoblast cells was determined using an actin immunofluorescence assay. Cellular morphology was observed using fluorescent microscopy and the relative cell size in responses to different administrations were analyzed using Zeiss Axio Vision software. The bar graph represents three independent experiments in which 60 cells were counted per condition in each experiment. Bars represent the relative cellularsize to control and indicated mean values ± S.D. (n=3).*=P <0.05,** =P <0.01 versuscontrol; #= P
<0.05, ##= P <0.01 versusLeu27IGF-II.
Fig. 4.Tanshinone IIA inhibited pathologic hypertrophy markers ANP and BNP on Leu27IGF-II-induced H9c2 cardiomyoblast cells and neonatal cardiomyocytes
hypertrophy. The cells were pretreated with ICI 182,780 (2 μM) for 1 h, followed by incubation with tanshinone IIA (10 and 100 nM) for 2 h; they were then treated with Leu27IGF-II (10 nM) for 24 h. (A) ANP and BNP protein levels in H9c2 cardiomyoblast cells were determined using western blot analysis. Equal loadswere verified with an anti-α-tubulin antibody.(B) RT-PCR was performed on RNA samples extracted from neonatal cardiomyocytes, using ANP- and IGF-IIR-specific primers.
The PCR products were electrophoresed on a 1.2% agarose gel stained with ethidium bromide. Quantitative analysis was performed with Gel-Pro Analyzer 4.0. Results are shown in mean values ± S.D. (n = 3). *= P <0.05,**= P <0.01 versuscontrol, #= P
<0.05, ##= P <0.01 versusLeu27IGF-II.
Fig. 5. Tanshinone IIA inhibited Leu27IGF-II-induced neonatal
cardiomyocyteshypertrophy through calcineurin-NFAT3 signaling.Neonatal
cardiomyocytes were incubated with tanshinone IIA and CsA (calcineurin inhibitor)
in the presence of Leu27IGF-II for 24 h. (A) NFATc3 and calcineurin protein levels
in neonatal cardiomyocytes were determined using western blot analysis. (B) ANP
and BNP protein levels in neonatal cardiomyocytes were determined using western
blot analysis. Equal loadswere verified with an anti-β-actin antibody.Quantitative
analysis was performed with Gel-Pro Analyzer 4.0. Results are shown in mean values
± S.D. (n = 3). *=P <0.05,**= P <0.01 versuscontrol, #= P <0.05, ##= P <0.01
versusLeu27IGF-II.
Fig. 6. Tanshinone IIA inhibited the NFAT3 nuclear localization on Leu27IGF-II-
induced H9c2 cell hypertrophy. The cells were pretreated with ICI 182,780 (2 μM) for
1 h, incubated with tanshinone IIA (10 and 100 nM) for 2 h, and then treated with
Leu27IGF-II (10 nM) for 24 h. (A) Immunofluorescence staining with specific
antibody against NFAT3 was performed and the results were visualized with a
fluorescence microscope coupled with an image analysis system. (B) Western blot
analysis for NFAT3 and calcineurin. Equal loadswere verified with an anti-α-tubulin
antibody.Quantitative analysis was performed with Gel-Pro Analyzer 4.0. Results are
shown in mean values ± S.D. (n = 3). *= P <0.05,**= P <0.01 versuscontrol, #= P
<0.05, ##= P <0.01 versusLeu27IGF-II.
Fig. 7.Tanshinone IIA activate survival pathway on Leu27IGF-II-induced H9c2 cells.
(A) H9c2 cells were pretreated with ICI 182,780 (2 μM) for 1 h, incubated with
tanshinone IIA 10 and 100 nM for 10 min, and harvested for the following analyses.
Cells were harvested and analyzed usingwestern blot using antibodies against p-Akt
and Akt. (B) H9c2 cells were pretreated with Ly294002 (5 μM) for 1 h, incubated
with tanshinone IIA 10 and 100 nM for 2 h, and then treated with (10 nM) of
Leu27IGF-II for 24 h. Cells were harvested and analyzed usingwestern blot using
antibodies against calcineurin, NFAT3, and BNP. Quantitative analysis was
performed with Gel-Pro Analyzer 4.0. Results are shown in mean values ± S.D. (n =
3). *= P <0.05,**= P <0.01 versuscontrol, #= P <0.05, ##= P <0.01 versusLeu27IGF-
II.
Fig. 8.Detection of PI3K siRNA inhibits the cardioprotective effects of tanshinone
IIA in Leu27IGF-II-treated primary cardiomyocytes.(A) Thirty-six hours after being
transfected with siRNA (100 nM non-targeting siRNA or PI3K siRNA) and
tanshionone IIA treatment for 20 min. Cells were harvested and Western blot analysis
was performed with antibodies against proteins as indicated.(B) Thirty-six hours after
being transfected with siRNA (100 nM non-targeting siRNA or PI3K siRNA),
neonatal cardiomyocytes were incubated with tanshinone IIA (100 nM) in the
presence of Leu27IGF-II for 24 h. The immunofluorescence staining with specific
antibody against NF-AT3 was performed and visualized with a fluorescence
microscope coupled with an image analysis system.(C) Neonatal cardiomyocyteswere
determined using an actin immunofluorescence assay. Cellular morphology was
observed using fluorescent microscopy, and the relative cell size in response to
different administration was analyzed using Zeiss Axio Vision software. The bar
graph represents three independent experiments in which 60 cells were counted per
condition in each experiment. Bars represent the relative cellularsize to control and
indicated mean values ± S.D. (n=3).*= P<0.05,**= P <0.01 versuscontrol, #= P
<0.05, ##= P <0.01versusLeu27IGF-II.
Fig. 9.A schematic representation showing that tanshinone IIA inhibits Leu27IGF-II-
induced IGF-IIR signaling and cardiomyocyte hypertrophy through activation of Akt.
Insulin-like growth factor (IGF-IIR) activation by specific Leu27IGF-II binding
induces IGF-IIR activation, which then leads to calcineurin activation. Calcineurin
then directly dephosphorylates NFAT3 transcription factor in the cytoplasm,
permitting translocation to the nucleus where dephosphorylated NFAT3 further
interacts with transcription factor (GATA-4) to form the complex that participates in
the development of myocardial hypertrophy and the hypertrophy response genes
expression such as ANP and BNP. Tanshinone IIA could block Leu27IGF-II-induced
apoptosis,and ICI administration totally reversed the tanshinone IIA effect.
Tanshinone IIA is therefore mediated through estrogen receptors inhibiting
Leu27IGF-II-induced calcineurin activation by specifically activating the PI3K-Akt
pathway, thereby inhibiting cardiomyocyte hypertrophy.
Figure 1
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Figure 5 (A)
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Figure 6 (A)
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Figure 7
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Figure 8 (A)
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Figure 9
